Differential Gene Expression and Withanolides Biosynthesis During in vitro and ex vitro Growth of Withania somnifera (L.) Dunal.

Ashwagandha (Withania somnifera L. Dunal) is a medicinally important plant with withanolides as its major bioactive compounds, abundant in the roots and leaves. We examined the influence of plant growth regulators (PGRs) on direct organogenesis, adventitious root development, withanolide biosynthetic pathway gene expression, withanolide contents, and metabolites during vegetative and reproductive growth phases under in vitro and ex vitro conditions. The highest shooting responses were observed with 6-benzylaminopurine (BAP) (2.0 mg L-1) + Kinetin (KIN) (1.5 mg L-1) supplementation. Furthermore, BAP (2.0 mg L-1) + KIN (1.5 mg L-1) + gibberellic acid (GA3) (0.5 mg L-1) exhibited better elongation responses with in vitro flowering. Half-strength MS medium with indole-3-butyric acid (IBA) (1.5 mg L-1) exhibited the highest rooting responses and IBA (1.0 mg L-1) with highest fruits, and overall biomass. Higher contents of withaferin A (WFA) [∼8.2 mg g-1 dry weight (DW)] were detected in the reproductive phase, whereas substantially lower WFA contents (∼1.10 mg g-1 DW) were detected in the vegetative phase. Cycloartenol synthase (CAS) (P = 0.0025), sterol methyltransferase (SMT) (P = 0.0059), and 1-deoxy-D-xylulose-5-phosphate reductase (DXR) (P = 0.0375) genes resulted in a significant fold change in expression during the reproductive phase. The liquid chromatography-mass spectrometry (LC-MS) analysis revealed metabolites that were common (177) and distinct in reproductive (218) and vegetative (167) phases. Adventitious roots cultured using varying concentrations of indole-3-acetic acid (IAA) (0.5 mg L-1) + IBA (1.0 mg L-1) + GA3 (0.2 mg L-1) exhibited the highest biomass, and IAA (0.5 mg L-1) + IBA (1.0 mg L-1) exhibited the highest withanolides content. Overall, our findings demonstrate the peculiarity of withanolide biosynthesis during distinct growth phases, which is relevant for the large-scale production of withanolides.

He-Ne laser accelerates seed germination by modulating growth hormones and reprogramming metabolism in brinjal.

A plant's ability to maximize seed germination, growth, and photosynthetic productivity depends on its aptitude to sense, evaluate, and respond to the quality, quantity, and direction of the light. Among diverse colors of light possessing different wavelengths and red light shown to have a high impact on the photosynthetic and growth responses of the plants. The use of artificial light sources where the quality, intensity, and duration of exposure can be controlled would be an efficient method to increase the efficiency of the crop plants. The coherent, collimated, and monochromatic properties of laser light sources enabled as biostimulator compared to the normal light. The present study was attempted to use the potential role of the He-Ne laser as a bio-stimulator device to improve the germination and growth of brinjal and to investigate the possible interactions of plant and laser photons. A substantial enhancement was observed in germination index, germination time and seed vigor index of laser-irradiated than control groups. The enhanced germination rate was correlated with higher GA content and its biosynthetic genes whereas decreased ABA content and its catabolic genes and GA/ABA ratio were noted in laser-irradiated groups during seed germination than control groups. Further the expression of phytochrome gene transcripts, PhyA and PhyB1 were upregulated in laser-irradiated seedlings which correlate with enhanced seed germination than control. Elevated levels of primary metabolites were noted in the early stages of germination whereas modulation of secondary metabolites was observed in later growth. Consequently, significantly increased photosynthetic rate, stomatal conductance, and transpiration rate was perceived in laser-irradiated seedlings compare with control. The current study showed hormone and phytochrome-mediated mechanisms of seed germination in laser-irradiated groups along with the enhanced photosynthetic rate, primary and secondary metabolites.

Red laser-mediated alterations in seed germination, growth, pigments and withanolide content of Ashwagandha [Withania somnifera (L.) Dunal].

Withania somnifera (L.) Dunal, generally well-known as Ashwagandha, is part of Indian traditional medicinal systems like Ayurveda, Siddha, and Unani for over 3000 years for treating an array of disorders. The chief bioactive component of this plant is the withanolides, a group of C28-steroidal lactone triterpenoids. These compounds are present in very low concentrations and hence cell culture methods have been used to enhance their production. Low-level laser irradiation has been reported to have elicited the seed germination, agronomical characters, biosynthesis of bioactive compounds in some plants. Therefore, the objective of the study was to investigate the effect of red (He-Ne) laser irradiation on seed germination, growth characters, pigment contents and withanolide content in W. somnifera. The seeds were inoculated onto two different combinations of Murashige and Skoog (MS) media and incubated for germination. The highest germination percentage was observed in ½ MS with pH 6.5 and GA3 presoaking followed by ½ MS with different pH. Four different doses of Helium-Neon (He-Ne) laser (10, 15, 20 and 25 J/cm2) were used to irradiate the seeds at 632.8 nm and germinated in vitro on ½ MS with pH 6.5. The maximum germination percentage, 63.88% was noted from seeds irradiated with 25 J/cm2 (P = 0.04). The highest total length of 13.33 cm was observed in the seedlings irradiated with 25 J/cm2 groups (P = 0.008). The highest total chlorophyll content of 329.5 µg/g fresh weight (FW) was observed for seedlings irradiated with 15 J/cm2 (P = 0.02) and the highest carotenoid content of 49.6 µg/g FW was observed for 25 J/cm2 treated seedlings. Further, primary root length was measured and found to be highest (11.14 cm) in seedlings irradiated with 10 J/cm2 and the highest number of lateral roots were observed for 15 and 25 J/cm2 groups. The significant amount of Withanolide A (WA) 0.52 µg/g dry weight (DW) and 0.60 µg/g DW was noted in 15 (P = 0.01) and 20 J/cm2 (P = 0.002) groups, respectively than control. The present investigation thus reveals the positive impact of red laser on the germination of seeds, growth characters and withanolide contents under in vitro environment.

Untargeted metabolomics and DNA barcoding for discrimination of Phyllanthus species.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus species is extensively cultivated and used as edible fruits and herbal drugs. The Phyllanthus species are used extensively as ethnopharmacologically important materials in several countries, especially in Asia. Various Phyllanthus species are broadly used in the Ayurvedic system of medicine and deliberated as bitter, astringent, stomachic, diuretic, febrifuge, deobstruent, and antiseptic, and used for the treatment of digestive, genitourinary, respiratory, skin diseases, hepatopathy, jaundice, and renal calculus in India. Precise authentification of Phyllanthus species is a challenge due to morphological similarities and is important to avoid adulteration found in herbal drugs. Hence, there is a need to establish comprehensive methods for the identification of Phyllanthus species. AIM OF THE STUDY: In this study, we attempted to integrate untargeted metabolomics to identify species-specific metabolites with traditional phylogenetic analysis for identification and discrimination of nine Phyllanthus species. MATERIALS AND METHODS: Phyllanthus species such as P. acidus, P. amarus, P. debilis, P. emblica, P. virgatus, P. urinaria, P. lawii, P. myrtifolius, and P. reticulatus were collected. The liquid chromatography coupled mass spectrometry (LC-MS) was performed for untargeted metabolite profiling and MS/MS fragmentation analysis was performed for selected compounds. Further, the barcoding analysis was executed using plastid loci, rpoC1 to integrate with metabolite profiling data. RESULTS: The Principal Component Analysis (PCA) of leaf metabolites showed distinct clusters in different species. Through further analysis, we have also identified the qualitative and quantitative status of unique metabolites across the species, and the majority of the selected compounds were annotated. The metabolic fingerprinting and the hierarchical clustering indicated that though the P. deblis and P. virgatus are distantly related to each other, they are closely associated with their metabolic profiling. Similarly, P. myrtifolius and P. urinaria are closely related to each other with their metabolic fingerprints than the genetic alignment. Further, we performed barcoding with rpoC1 across nine Phyllanthus species (P. acidus, P. amarus, P. debilis, P. emblica, P. virgatus, P. urinaria, P. lawii, P. myrtifolius, and P. reticulatus). Sequence similarity search in the GenBank database showed rpoC1 barcode loci from nine Phyllanthus species showed significant identity (>97%) with the sequences of various Phyllanthus species. CONCLUSIONS: The bioactive metabolites and their abundance can be assigned to specific species thereby serving as a biological signature and indicators for potential therapeutic use. This study identified differential expression of 14 secondary metabolites from nine Phyllanthus species. Alkaloid compound zeatin was found specific to P. virgatus and delphinidin-3-O- ß -D-glucoside was not found in P. myrtifolius. Barcoding and phylogenetic analysis showed P. acidus is the most genetically distinct among the groups and the sequence pair between P.emblica-P.reticulatus and P.emblica-P.urinaria showed the least difference.

ROS-dependent DNA damage and repair during germination of NaCl primed seeds.

Reactive oxygen species (ROS) generated during rehydration of seeds is a major source of cellular damage. Successful germination depends on maintaining the oxidative window and ability of the cells to repair the DNA damage accumulated during seed developmental process, maturational drying, and germination. We explored the role of DNA damage, repair, cell cycle progression and antioxidant machinery in germination of seeds of Solanum melongena L. primed with 0, 320, 640 and 1200 mM sodium chloride (NaCl). The expression of antioxidant genes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase2 (CAT2), and glutathione reductase (GR) was upregulated to maintain the oxidative window required for germination in seeds treated with 320 mM NaCl. ROS generated upon treatment with 320 mM NaCl resulted in minimal DNA damage and activated non-homologous end joining (NHEJ) and mismatch repair (MMR) pathway genes such as KU70 and mutS homolog 2 (MSH2) respectively. Treatment with higher concentrations of NaCl resulted in increased DNA damage despite lower ROS, without evoking DNA repair mechanisms. Uncontrolled rehydration resulted in higher levels of ROS and DNA damage, but activation of homologous recombination (HR) pathway gene, Nijmegen breakage syndrome 1 (NBS1), and genes involved in repairing oxidized guanine, such as oxoguanine DNA glycosylase (OGG1) and proliferating cell nuclear antigen (PCNA). In summary, controlled rehydration with 320 mM NaCl decreased the DNA damage, reactivated the antioxidant and DNA repair machinery, and cell cycle progression, thereby enhancing the seed germination.

Responses of He-Ne laser irradiation on agronomical characters and chlorogenic acid content of brinjal (Solanum melongena L.) var. Mattu Gulla.

Exposure to laser irradiation on seeds brings about the changes in agronomical characteristics of the plants. Solanum melongena L. var. Mattu Gulla, a variety of brinjal is of high economic value due to its unique colour and flavour. The aim of the study was to understand the influence of Helium-Neon (He-Ne) laser irradiation on agronomical characters of Solanum melongena L. var. Mattu Gulla in the field conditions. Various growth characteristics including seed germination percentage, survival rate, plant height, number of branches, and flowers and fruits were estimated during different developmental stages of the brinjal. In addition, the chlorogenic acid content of fruits obtained from the laser irradiated seeds were quantified using reversed-phase high-performance liquid chromatography (RP- HPLC). The plants from the seeds irradiated with different doses (20, 25 and 30J/cm2) of He-Ne laser showed significant enhancement on the growth characteristics when compared to the non-irradiated control groups. He-Ne laser irradiation also improved the yield characteristics of the plants significantly in in vivo conditions in comparison with control group. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed using methanolic extract of matured fruit of Mattu Gulla on HepG2 and fibroblast cell lines. The IC50 values of fruit extract from laser irradiated groups were found to be similar to non-irradiated control groups. Chlorogenic acid content was found to be higher in 20J/cm2 and lower in 30J/cm2 treated fruit tissue. The current study thus elucidates the role of He-Ne laser as a biostimulator on brinjal var. Mattu Gulla not only in the in vitro conditions but also in the in vivo field conditions.